CYCLOGUO

"Formation and roles of the 5',8-cyclo-2'-deoxyguanosine lesion"

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 Obiettivo del progetto (Objective)

'Free radical attack to genetic material leads to damages that are involved in pathologies as well as in aging. Guanine is the preferred oxidation site of DNA, and 8-oxo-dG is used as marker of radical stress, with some drawbacks due to oxidative artefacts during the analysis. The project addresses the chemical biology of 5’,8-cyclo-2’-deoxyguanosine (5’,8-cyclo-dG), a tandem damage produced by free radical insult to the 5’ position of the sugar. The compound has an extra covalent bond between the base and sugar moieties and occurs as 5’S and 5’R diastereomers. Chemical studies on 5’,8-cyclo-dG, paralleled with 8-oxo-dG, are expected to contribute to a full scenario of guanine damage. The project addresses: i) Structure and reactivity features of the 5’,8-cyclo-dG formation and diastereomeric ratios as reporter of different DNA supramolecular organization and environmental factors; ii) chemical and stereochemical issues in different oligosequences and DNA-mimics, including convenient syntheses of various (5’S)- and (5’R)-5’,8-cyclo-dG structures, which represent the limiting step for comprehensive biological and medical research; iii) the 5’,8-cyclo-dG damage in guanine-rich regions such as telomeres, “CpG islands” and introns, intensively studied for cancer and aging processes; iv) aspects of the structural distortion of guanine-rich regions due to the lesion; v) differences in enzymatic recognition and hydrolysis of sequences containing the lesion in the two diastereomeric forms. Main milestones of the project are envisaged: easy access to cyclonucleoside-containing substrates; protocols for characterization of the 5’,8-cyclo-dG lesions and application to damage evaluation in biological samples; assessment of damage in different dietary conditions and cell environments, such as nuclear and mitochondrial DNA; balanced aspects of the damage and repair processes of the 5’,8-cyclo-dG lesion; integration of the 5’,8-cyclo-dG lesion in the whole scenario of DNA damage.'

Introduzione (Teaser)

Oxidative damage to genetic material affects its integrity and stability, and leads to development of pathologies. Chemical studies of DNA reactivity are fundamentally important as an individual cell can suffer up to 1 million DNA changes per day.

Descrizione progetto (Article)

The hydroxyl radical OH represents one of the most dangerous species that alter biomolecules and are involved in biological damages and ageing. This reactivity can cause changes of the four nucleotide units adenosine, guanosine, thymine and cytosine. The EU-funded 'Formation and roles of the 5',8-cyclo-2'-deoxyguanosine lesion' (CYCLOGUO) project specifically addressed the DNA lesions known as tandem damage of the purine bases 2'-deoxyadenosine and 2'-deoxyguanosine.

Project partners focused on chemical studies of the 5',8-cyclopurine lesions (5',8-cPus), solving problems in the synthesis of the four diastereomeric structures. The availability of the four lesions is necessary in order to conduct subsequent studies on their structural, functional and biological properties. The incorporation of these modified phosphoroamidite analogues in oligonucleotide sequences can simulate the lesions occurring on the DNA strands. This biomimetic study is necessary for understanding of the potential consequences in vivo.

The CYCLOGUO project dealt with the synthesis of the 5',8-cPus and their phosphoramidites derived from 2'-deoxyguanosine, and developed a synthetic protocol for the 5',8-cyclo-dGuo lesions. The new methodology was completed with the synthesis of 5',8-cyclo-dGuo phosphoramidites. The two adenine 5',8-cPu phosphoramidites were also synthesised and each lesion incorporated into oligonucleotides strands according to the DNA solid-phase synthesis technique.

Additionally, the 5',8-cPus compounds have been developed as DNA oxidative stress biomarkers for comparison with currently used 8-oxo-7,8-dihydro-2'-deoxyguanosine. Researchers synthesised the isotopic-enriched 15N equivalents of the four 5',8-cPus for the evaluation of DNA samples based on the stable isotope dilution tandem mass spectrometry technique.

CYCLOGUO studied the chemical biology of DNA damage and repair. This topic has significant medical application, since the alteration of DNA and impairment of the repair systems lead to mutations and diseases.